Jd. Phillips et al., EXPRESSION AND BIOCHEMICAL-CHARACTERIZATION OF IRON REGULATORY PROTEIN-1 AND PROTEIN-2 IN SACCHAROMYCES-CEREVISIAE, Biochemistry, 35(49), 1996, pp. 15704-15714
Iron-regulatory proteins (IRPs) 1 and 2 are cytosolic RNA-binding prot
eins that bind to specific stem-loop structures, termed iron-responsiv
e elements (TREs) that are located in the untranslated regions of spec
ific mRNAs encoding proteins involved in iron metabolism. The binding
of IRPs to IREs regulates either translation or stabilization of mRNA.
Although IRP1 and IRP2 are similar proteins in that they are ubiquito
usly expressed and are negatively regulated by iron, they are regulate
d by iron by different mechanisms. IRP1, the well-characterized IRP in
cells, is a dual-function protein exhibiting either aconitase activit
y when cellular iron is abundant or RNA-binding activity when cellular
iron is scarce. In contrast, IRP2 lacks detectable aconitase activity
and functions exclusively as an RNA-binding protein. To study and com
pare the biochemical characteristics of IRP1 and IRP2, we expressed wi
ld-type and mutant rat IRP1 and IRP2 in the yeast Saccharomyces cerevi
sine. IRP1 and IRP2 expressed in yeast bind the IRE RNA with high affi
nity, resulting in the inhibition of translation of an IRE-reporter mR
NA. Mutant IRP2s lacking a 73 amino acid domain unique to IRP2 and a m
utant IRP1 containing an insertion of this domain bound RNA, but lacke
d detectable aconitase activity, suggesting that the presence of this
domain prevents aconitase activity. Like LRP1, the RNA-binding activit
y of IRP2 was sensitive to inactivation by N-ethylmaleimide (NEM) or 5
,5'-dithiobis(2-nitrobenzoic acid) (DTNB), indicating IRP2 contains a
cysteine(s) that is (are) necessary for RNA binding. However, unlike I
RP1, where reconstitution of the 4Fe-4S cluster resulted in a loss in
RNA-binding activity, the RNA-binding activity of IRP2 was unaffected
using the same iron treatment. These data suggested that IRP2 does not
contain a 4Fe-4S cluster similar to the cluster in IRP1, indicating t
hat they sense iron by different mechanisms.